Biology subjects

Bjelcic, M.

Publications and source records attributed to Bjelcic, M..

4 recordsLinked to original sources

bioRxiv2026

Photoactivatable Rac1: a photosensor tilt by time-resolved crystallography and a partially open switch I when GDP-bound

The LOV2 domain of Avena sativa phototropin 1 is widely used to control proteins with light, but how its signal reaches a fused effector has not been visualized. We followed photoactivatable Rac1 by time-resolved serial crystallography at room temperature. Within 350 {micro}s, a fifth of the photosensors form the flavin-cysteine adduct and tilt as a rigid body on Rac1 about an axis through the domain interface, while the J helix stays docked and Rac1 keeps GTP and dark-state switches up to 1.85 ms. Dark crystals contain a variable GDP-bound sub-population, so light data must be referenced to interleaved dark frames. In this sub-population, switch I is only partially open, held at a Ca2+ ion that bridges LOV2 and Rac1, a conformation that can persist in simulations without the lattice. The photosensor thus first acts on Rac1 at the interface that also holds switch I in the GDP-bound state.

biophysics↗

bioRxiv2026

Anaerobic time-resolved serial crystallography captures CO dissociation and rebinding in oxygen-sensitive -hydrogenase

Redox-active, oxygen-sensitive metalloenzymes catalyze key reactions in biological energy conversion and small-molecule activation. Understanding their mechanisms requires structural characterization of transient catalytic intermediates. Time-resolved serial crystallography (TR-SX) enables direct visualization of protein dynamics during catalysis under near-physiological conditions. Its application to oxygen-sensitive enzymes has remained challenging because strict anaerobic conditions must be maintained throughout sample preparation and data collection. Here, we establish an anaerobic room-temperature serial crystallography workflow for the oxygen-sensitive [FeFe]-hydrogenase CpI and demonstrate its applicability by determining a room-temperature structure of the CO-inhibited Hox-CO state and following inhibitory CO dissociation and rebinding on the millisecond timescale via TR-SX. The presented room-temperature Hox-CO structure closely resembles previous cryogenic models and shows no detectable evidence of oxygen-induced degradation or significant radiation damage. Time-resolved measurements reveal no detectable structural rearrangements accompanying CO dissociation and rebinding beyond displacement and return of the inhibitory ligand, indicating a rigid catalytic architecture that may facilitate rapid catalysis. The presented workflow enables time-resolved structural studies of oxygen-sensitive metalloenzymes under physiologically relevant conditions and opens the way to direct visualization of catalytic intermediates in redox enzymes.

biochemistry↗

bioRxiv2025

A user-friendly goniometer-compatible fixed-target platform formacromolecular crystallography at synchrotrons

Fixed-target platforms provide convenient support for microcrystals during serial X-ray crystallography studies using synchrotron radiation. Here, we describe a simple, user-friendly 3D-printed support where the crystals are sandwiched between two layers of thin X-ray transparent membrane resulting in very low scattering background. The platform is compatible with magnetic mounting onto the standard goniometer of macromolecular crystallography beamlines. Our design utilizes a 96-well frame that facilitates hanging-drop experiments directly on the membrane using conventional crystallization plates, thereby eliminating multiple pipetting and crystal handling steps. Crystals can be enclosed into a sandwich and packed into cassettes, preventing the risk of the sample drying out during room-temperature transportation to synchrotron sources. The versatility of the platform is demonstrated by five structures solved using different crystallization- and data-collection strategies. Single crystal rotational-crystallography at both room- and cryogenic-temperatures using large crystals of lysozyme is shown. On-chip microcrystallization is illustrated by use of a photosynthetic reaction center as an example. Finally, serial crystallography data collection at room-temperature from microcrystals of photosynthetic reaction center as well as cytochrome c oxidase crystallized in lipidic cubic phase is presented.

biophysics↗

bioRxiv2023

Exploring serial crystallography for drug discovery

Structure-based drug design is highly dependent on the availability of structures of the protein of interest in complex with lead compounds. Ideally, this information can be used to guide the chemical optimization of a compound into a pharmaceutical drug candidate. A limitation of the main structural method used today, rotational cryo-crystallography, is that it only provides structural information of the protein-complex in its frozen state. Serial crystallography is a relatively new approach that offers the possibility to study protein structures at room-temperature. Here, we explore the use of serial crystallography to determine structures of the pharmaceutical target soluble epoxide hydrolase. We introduce a new method to screen for optimal microcrystallization conditions suitable for use in serial crystallography and present a number of room-temperature ligand-bound structures of our target protein. From a comparison between the room-temperature structural data and previously published cryo-temperature structures, we describe an example of a temperature-dependent difference in ligand-binding mode and observe that flexible loops are better resolved at ambient temperature. Finally, we discuss current limitations and potential future advances of serial crystallography for use within pharmaceutical drug discovery.

biochemistry↗